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 Ethylene Glycol Screening: Adapting the CATACHEM Enzymatic Assay to Minimize False Positives Lindsay Ethylene Glycol Screening: Adapting the CATACHEM Enzymatic Assay to Minimize False Positives Lindsay Hardy 1, Shu-Ling Fan 1, Adele Pistorino 1, Jo. Etta Juenke 2, Gwendolyn Mc. Millin 3, Gary Horowitz 1 1 Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA. , 2 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA, 3 Department of Pathology, University of Utah, Salt Lake City, UT, USA. RESULTS INTRODUCTION: Ethylene glycol (ETG) is a toxic substance found in automotive anti-freeze. The most commonly used methods for quantification are gas chromatography (GC) with flame-ionization (FID) or mass spectrometry (MS). A veterinary medicine enzymatic assay, by CATACHEM, was previously noted for interfering substances, including propylene glycol (PG) and 1, 3 -butanediol (BD). OBJECTIVE: In this study, new method parameters for the CATACHEM assay were selected in an effort to minimize interferences and other sources of false positive results. RESULTS: 1) Recovery studies for both methods were linear as high as 250 mg/d. L. Accuracy and precision tests revealed CV of: 3. 4% at 61. 7 mg/d. L, 1. 2% at 227. 9 mg/d. L. 2) Three analytes were tested for interference: glycerol, PG, and BD. Glycerol did not interfere with either method. PG and BD gave falsely elevated ETG results at all concentrations using the original method; neither compound gave falsely elevated ETG readings with the new method. 3) Twenty de-identified clinical samples with ethanol ordered were tested. Using the original method, five yielded ETG levels >10 mg/d. L. Using the new method, all twenty samples were reported as <10 mg/d. L of ETG. 4) For 13 clinical samples, the modified data reduction yielded 3 results with error messages, two of which were beyond the assay’s measuring range (~500 mg/d. L). Compared to GC-FID, for the 10 samples without error messages, r-squared = 0. 97. Kinetics Interfering Substances (BIDMC) Two Point End: Compares the absorbance at two time points Rate A: Measures the slope of the increased absorbance over many points Identity Original Method New Method Ethylene Glycol 236 mg/d. L 226 mg/d. L 245 mg/d. L Propylene Glycol 20 mg/d. L 61 mg/d. L <10 mg/d. L Sample Identity Ethylene Glycol (mg/d. L) BIDMC # AU 400 CATACHEM ARUP Ethanol (mg/d. L) Original Method New Method 1 0 15 <10 71 31 <10 73 18 <10 Assay/Time/Point [2 Point End] [17] [31] [Rate A] [17] [27] Wavelength 1 340 Wavelength [405] [340] Wavelength 2 410 2 Absorbance Limit [0] [Increase] [6000] [Increase] Method Rate Fixed 3 [-99999] [99999] 69. 6 *Lin 273 319. 5 *Lin 81 25. 6 *Lin 95 34. 2 *Lin 68 Reaction + + 4 182 18 <10 Point 1 First 11 0 Point 1 Last 27 16 5 261 10 <10 700 600 500 400 300 200 100 0 -100 600 500 400 300 200 100 300 500 700 900 24. 5 *Lin 0 -100 400 900 Gas Chromatography – Flame Ionization Detector (mg/d. L) Instrument 367 2710. 6 *Limt 1 52 22. 9 39 n AU 400 (ARUP) 32 y = 1. 04 x - 2. 94 r 2 = 0. 98 Hitachi 917 (BIDMC) 17 y = 1. 01 x - 2. 15 r 2 = 0. 99 21 Precision Data • In the presence of interfering substances, the new method generated either accurate results or flagged an error message, whereas the old method simply generated (erroneous) elevated values. ethylene glycol, formic acid, isopropanol, methanol, glycolic acid, oxalic acid, glyoxylic acid, 1, 2 butanediol, 1 butanol, 1, 4 butanediol, 800 Gas Chromatography – Flame Ionization Detector (mg/d. L) • Overall, 67 samples were spiked either in combination or alone with: Ethylene Glycol Parameters for Two Instruments [-99999] [99999] 118 50 mg/d. L PG Glycolic acid 50 mg/d. L ETG Isopropanol 20 mg/d. L ETG 30 mg/d. L 1, 2 BD 20 mg/d. L ETG 150 mg/d. L PG 20 mg/d. L ETG 20 mg/d. L PG 20 mg/d. L ETG 8 mg/d. L 1, 2 BD Elimination of False Positives in Ethanol Samples Sensitivity Limit 97. 8 *Lin 2, 3 BD NADH + H+ BIDMC 217 1, 2 BD Glycoaldehyde CATACHEM 29. 5 *Lin 100 mg/d. L PG Buffer, Stabilizer Nonreactive ingredients Glycerol Dehydrogenase Hitachi. TM 917 60 800 New Method (mg/d. L) 27 mg/d. L ETG (Patient sample) METHOD: A bacterial enzyme, Glycerol Dehydrogenase, oxidizes ETG in the presence of NAD, causing the production of NADH and an increase in absorbance at 340 nm. NAD+ Original Method (mg/d. L) Identity 900 Enzymatic Method (mg/d. L) 900 METHODS Ethylene Glycol Patient Correlation Enzymatic Method (mg/d. L) ABSTRACT propylene glycol, n-propanol, acetone, ethanol, polyethylene glycol, glyoxal solution, 1, 2 butanediol, 1, 3 propanediol, 1, 3 butanediol, 1 -octanol • In no case did the new method generate an erroneous result. ETG (mg/d. L) 11 40 56 100 248 Within Run CV (%) 1. 48 2. 02 1. 75 1. 6 1. 12 n 20 20 20 Between Run CV over 5 Days (%) 0. 35 0. 19 3. 49 0. 19 7. 55 n 20 20 20 CONCLUSIONS v The new method exhibits excellent agreement with the reference method (gas chromatography – flame ionization detection). v In contrast to the original method, no false positive ethylene glycol results were generated among the samples tested (patient and spiked). v The new method was successfully implemented at two sites, using two different instruments, with excellent agreement. v This new method may make it practical for many laboratories to offer accurate ethylene glycol determinations on routine chemistry instrumentation.